16.5 Entomogenous fungi

Entomogenous means ‘growing on or in the bodies of insects’ and
strictly-speaking applies to all those organisms already discussed in this
Chapter. However, the word is most often used to describe filamentous fungi that
invade their insect hosts by penetrating directly through the
cuticle, whereas most organisms in the groups we have already described are
either ectoparasites or insect pathogens that generally infect the host when
infective spores are ingested into the intestine.

In entomogenous (or ‘entomopathogenic’)
fungi (Table 1) the general infection cycle is that a fungal spore adheres to
the cuticle and germinates when conditions are suitable.

Table 1. Examples of entomogenous fungi

Fungus

Taxonomic position

Host

Coelomomycespsorophorae

Blastocladiomycota

Alternates between the larvae of the mosquito (Culiseta inornata)
and the copepod, Cyclops vernalis

Erynia

Zygomycota: order Entomophthorales

Erynia neoaphidis is a pathogen of aphids; Erynia radicans
has potential to biocontrol the eastern spruce budworm

Entomophthora muscae

Zygomycota: order Entomophthorales

Adult Diptera, including houseflies

Verticillium lecanii now called Lecanicillium lecanii

Ascomycota: Hypocreales

Aphid, whitefly, used in production of Vertalec (used against glasshouse
aphid) and Mycotal (used against glasshouse whitefly).

The germ tube of the spore then penetrates the host cuticle with a
combination of enzymes and physical force and enters the body cavity (haemocoel
or haemolymph) of the insect host (diagrammed in Fig. 7, with an indication of
the insect defences) (Butt et al., 2016).

Fig. 7. Schematic representation of infection structures of
Metarhizium anisopliae (Ascomycota). This is a fungus, which is found
in soils throughout the world, that causes disease in over 200 insect
species. The illustration shows penetration of the insect cuticle in a
diagrammatic cross-section. Cuticular resistance barriers may be (a)
preformed (including those at the surface, like hydrophobicity,
electrostatic charges, other microbes on the surface, low relative humidity
and low nutrient availability, toxic lipids and phenols, and/or tanned
proteins in the epicuticle; and tanned proteins with crystalline chitin to
stiffen and dessicate the procuticle, and protease inhibitors in the
procuticle), and/or (b) induced, such as melanisation which cross links
cuticle components to provide a measure of resistance to fungal hydrolytic
enzymes. Based on a figure in Hajek & St. Leger, 1994.

Within the body cavity the fungus grows vegetatively, often in the form of
yeast-like hyphal bodies that reproduce by budding, eventually
filling the haemocoel and sending hyphae into the solid tissues of the host. As
a result, the fungus depletes the tissues of nutrients and the host suffers
starvation and may also be poisoned by toxins produced by the fungus.
Eventually, the insect dies and the host cuticle ruptures (see the flow-chart
summary in Fig. 8).

Fig. 9. The final stages of Beauveria bassiana (Ascomycota)
infections. A, B and C
show the effect of B. bassiana infection on whiteflies,
Trialeurodes vaporariorum. A shows the lower surface
of a leaf heavily infested with whitefly on which many pupae have been
infected by Beauveria (circled region in particular). B
is a magnified view of the leaf surface showing a comparison between an
infected whitefly pupa and a healthy one. The infected pupa is red due to
the accumulation of large amounts of the red antibiotic oosporein, which is
the major secondary metabolite excreted by Beauveria and several
other fungi. Oosporein is an antifungal agent; is also antagonistic to
Phytophthora infestans, and is a mycotoxin that causes skeletal
problems in poultry fed contaminated grain. C is another
whitefly cadaver showing conidiospore production. D is a
cadaver of Dicyphus hesperus, which is used as a predator in
biocontrol of whiteflies and spider mites in greenhouses but is also
sensitive to Beauveria infection. Photographs by Roselyne Labbé.
Modified from Labbé, R. (2005). Intraguild interactions of the
greenhouse whitefly natural enemies, predator Dicyphus hesperus,
pathogen Beauveria bassiana and parasitoid Encarsia formosa.
M.Sc. thesis, Faculté des Sciences de L’Agriculture et de L’Alimentation,
Université Laval, Canada at
http://archimede.bibl.ulaval.ca/archimede/files/4ee0acbe-a906-4104-9589-3ee49c48d09b/22512.html,
and see Labbé et al., 2009. Photographs kindly supplied by Dr
Roselyne M. Labbé, Agriculture and Agri-Food Canada.

This final stage is not seen in a few fungal pathogens that rely on dispersal
by insect flight or other movement of the infected hosts, for example
Massospora (Entomophthorales, ‘Zygomycota’), which causes a fungal disease
that affects the Cicada, and Strongwellsea spp. (Entomophthorales,
‘Zygomycota’) which are common fungal pathogens of different types of adult
flies. However, an insect cadaver covered by fungal mycelium from which fruit
bodies are emerging and/or around which lays a carpet of released spores is the
stage most commonly observed in the field (view the video in the
Resources Box). Because they are easily visible to the naked eye,
observation of these final stages allowed early observations of fungal disease
in commercially-valuable insects like the honey bee and silkworm, and helped
give birth to invertebrate pathology as a field of study.

Entomogenous fungi have been described from all the major fungal
phyla: chytrids, zygomycetes, Ascomycota, and Basidiomycota and over 700 species
of fungi are known to infest insects. The zygomycetes and Ascomycota contain
some extremely common insect pathogens that are also useful in
biocontrol programmes (see next section, below). Although some fungi infect a
range of insects (for example, Verticillium lecanii infects aphids,
thrips and whitefly), other species can be extremely specific (Erynia
neoaphidis only infects aphids). The infection process frequently involves
specific fungus-insect host recognition interactions that can even be
strain-specific for both fungus and host
(Wraight et al., 2007) .

For example, two closely related species of Coelomomyces
(Blastocladiomycota), C. dodgei and C. punctatus, have
sporangia with similar sizes, shapes, and general morphology, and their host
(mosquito) species ranges and geographic distributions overlap; but they do not
interbreed and experimentally-forced hybrid zygotes were only partially viable,
giving biological support to their classification into two distinct
morphospecies (Castrillo et al., 2005).

The initial attachment step can be non-specific, involving passive
hydrophobic interactions between the insect cuticle and hydrophobin
proteins on the surface of the fungal spores (see the section entitled On
the far side in Chapter 6; CLICK
HERE to view the page). Spores of the common entomogenous fungi
Beauveria bassiana, Metarhizium anisopliae and Nomuraea rileyi
are like this and will adhere to both host and non-host insects.

A mucilaginous coat also permits passive attachment to
surfaces in members of the Entomophthorales. In some aquatic entomogenous fungi
initial contact between fungal zoospores and mosquito larvae is due to the
zoospores having a negative geotaxis that favours collisions with mosquito
larvae near the water surface. In contrast, selective attachment depends on the
spore (or germ tube) interpreting chemical and physical cues on the host
cuticle; this applies to the attachment to mosquito larvae of some species of
the chytrid Coelomomyces.

In terrestrial filamentous fungi, spore germination results in the emergence
of a penetrating germ tube, or a germ tube and appressorium. In either case a
narrow penetration peg then breaches the insect cuticle using
mechanical (turgor pressure) and/or enzymic means, particularly proteases in the
latter case because insect cuticle comprises up to 70% protein, but including
chitinases and lipases (Charnley, 2003; Pereira et al., 2007). Many
spores also secrete mucilage during the formation of infective structures that
enhances adhesion to the host cuticle. Appressorium formation
and production of cuticle-degrading proteases are triggered by low nutrient
levels in M. anisopliae, demonstrating that the fungus senses
environmental conditions and host cues at the beginning of the infection
process. The enzymes are produced in sequence; protease followed by chitinase is
needed to dissolve the insect cuticle. An important point is that
cuticle-degrading enzymes not only aid penetration but also provide the fungal
germ tube with nutrients (Castrillo et al., 2005).

Entomogenous fungi are dimorphic (see the section entitled
Yeast-mycelial dimorphism in Chapter 5;
CLICK HERE to view the page) and after penetration the fungus changes from
filamentous hyphal growth to growing as yeast-like or protoplast hyphal bodies
that circulate in the haemolymph and multiply by budding. Later, the fungus
changes back to a filamentous mode of growth to invade internal tissues and
organs. In Entomophthora hyphal growth morphology was induced by
low-nutrient conditions, particularly low nitrogen availability (Castrillo
et al., 2005). The value of their filamentous morphology to entomogenous
fungi is worthy of specific comment. Fungal pathogens of plants and insects have
evolved similar methods of breeching the surfaces of their hosts, that is, they
both form appressoria from which narrow hyphae penetrate the host’s surfaces by
a combination of mechanical pressure and enzymatic softening. Filamentous fungi
are far more successful than unicellular bacteria as pathogens of plants; and
the same is true for insects. The reverse applies to their comparative success
as pathogens of higher animals.

During growth in the host the fungi also produce a wide variety of
toxic metabolites, which vary from low molecular weight products of
secondary metabolism to complex cyclic peptides and enzymes, some of which are
insecticidal. Few compounds have been found in diseased insects in sufficient
quantity to account for disease symptoms. An exception is a family of cyclic
peptides called the destruxins. These are cyclic depsipeptide
toxins from Metarhizium spp. (a depsipeptide is a peptide in
which one or more of the amide (-CONHR-) bonds are replaced by ester (-COOR)
bonds). Twenty-eight different destruxins have been described with different
levels of activity against different insects. The amount of destruxin produced
correlates with virulence and host specificity. Destruxins modulate the host
immune system and inhibit phagocytes (Charnley, 2003; Liu & Tzeng, 2012).

Many other toxins are produced by entomopathogenic fungi; for example, the
entomogenous fungus Entomophthora muscae, which infects and kills
domestic flies (Musca domestica), produces pheromones
which
attract other flies, especially males, towards dead, infected flies
producing Entomophthora spores: come hither and be infected is
the fungus-modified message! Some of the fungal secondary metabolites must be
neurotoxins because changes in insect behaviour are a common feature of fungal
infections. In particular, the infected host is frequently driven to
climb up vertical surfaces in the final stages of its life: this might
mean up climbing up plant stems, rocks, or to the top of walls and windows in
domestic pest insects. Being located at altitude is an advantage for spore
distribution by the fungus, but it is the dying, infected insect that does the
climbing.

Eventually, the fungus emerges through the cuticle and an external mycelium
grows over the host and, in appropriate environmental conditions, produces the
sporing structures. If circumstances are not suitable, for example dry and/or
cold, some fungi form resting structures inside the insect cadaver that produce
infective spores when conditions improve.